Human platelet-derived growth factor (PDGF) has been shown to be the major mitogenic protein in serum for mesenchymal-derived cells. This is well documented by numerous studies showing induction of either cell multiplication or DNA synthesis (a prereguisite for cell division) in cultured smooth muscle cells, fibroblasts and glial cells by platelet-rich plasma or purified PDGF (Ross et al., Proc. Natl. Acad. Sci USA 71:1207, 1974; Kohler and Lipton, Exp. Cell Res. 87:297, 1974; Westermark and Wasteson, Exp. Cell. Res. 98:170, 1976; Heldin et al., J. Cell Physiol. 105: 235, 1980; Raines and Ross, J. Biol. Chem. 257:5154, 1982). Furthermore, PDGF is a potent chemoattractant for monocytes and for cells that are responsive to it as a mitogen (Grotendorst et al., J. Cell Physiol. 113:261, 1982; Seppa et al., J. Cell Biol. 92: 584, 1982). PDGF has also been reported to be a chemoattractant for neutrophils. Due to its mitogenic activity, PDGF is useful as a component of a defined medium for the growth of mammalian cells in culture and as a research reagent with multiple applications in the study of animal cell biology.
In vivo, PDGF normally circulates stored in the alpha granules of platelets. Injury to arterial endothelial linings causes platelets to adhere to the exposed connective tissue and release their granules. The released PDGF is thought to chemotactically attract fibroblasts, smooth muscle cells and monocytes/macrophages to the site of injury and to induce the focal proliferation of fibroblasts and smooth muscle cells as part of the process of wound repair (Ross and Glomset, N. Eng. J. of Med. 295:369, 1976). PDGF is also produced by a number of other cell types, including endothelial cells.
PDGF has been demonstrated to be an effective wound-healing agent in several animal models of wound healing (Murray et al., U.S. patent application Ser. No. 07/230,190; Thomason et al., EP 282,317; Greenhalgh et al., Am. J. Pathol. 136: 1235-1246, 1990) and has been used in combination with insulin-like growth factor 1 (IGF-1) to promote bone healing (U.S. Pat. No. 4,861,757) and in combination with transforming growth factor alpha (U.S. Pat. No. 4,874,746).
It has been postulated that as a part of the response to injury of the arterial wall, PDGF released by platelets may play a causative role in the development of the proliferative lesions of atherosclerosis (Ross and Glomset, ibid.), which is one of the principal causes of myocardial and cerebral infarction.
Natural PDGF may be isolated from human plasma or platelets as starting material, but this is a complex and expensive process, in part due to the limited availability of the starting material. In addition, it is laborious to purify PDGF by classical methods at a high yield from other serum components due to its extremely low abundance and biochemical properties. Furthermore, the therapeutic use of products derived from human blood carries the risk of disease transmission due to contamination by, for example, hepatitis virus, cytomegalovirus, or HIV.
PDGF can now be produced by recombinant DNA techniques (U.S. Pat. Nos. 4,766,073; 4,769,328; 4,801,542; 4,845,075 and 4,849,407), thus overcoming the cost and risk of contamination associated with its isolation from plasma or platelets. However, both the native and recombinant forms of PDGF exhibit amino-terminal sequence heterogeneity, indicating that the molecule is sensitive to proteolysis. Such heterogeneity can interfere with product uniformity and may therefore be undesirable in a therapeutic compound.
In view of PDGF's clinical applicability in the treatment of injuries in which healing requires the chemoattraction and proliferation of fibroblasts or smooth muscle cells and its value as an important component of a defined medium for the growth of mammalian cells in culture, the production of useful quantities of protein molecules with activities comparable to those of native PDGF is clearly invaluable. There is therefore a need in the art for compositions of biologically active PDGF-like proteins that are resistant to proteolysis and therefore more homogeneous. The present invention provides such proteins and further provides other, related advantages.